This invention relates to Raman Spectrometers. In particular, it relates to phase sensitive detection of Raman scattering.
Raman spectroscopy (RS) is a useful tool for studying the structure of solids. However, Raman scattering (RS) is often swamped by fluorescence from resonantly excited molecules under study or from other sample constituents including impurities. In particular cases, fluorescence may be reduced by judicious choice of exciting frequency, careful sample preparation or the introduction of fluorescence quenching molecules or surfaces. However, in order to fully exploit resonant RS and to study inhomogeneous systems such as, for example, in vivo biological samples or molecules on dispersed supports, a general means of fluorescence suppression is required. Numerous approaches using both stimulated and spontaneous RS have been developed to increase the contrast between RS and fluorescence for particular samples.
Nonlinear optical techniques such as coherent anti-stokes RS (CARS) or Raman gain spectroscopy in which the frequency difference between two exciting lasers is scanned have yielded fluorescent free Raman spectra of transparent samples. Interference due to nonresonant terms often complicate the CARS spectrum, but these can be suppressed using Raman-induced Kerr effect spectroscopy (RIKES). A host of linear methods which separate RS from fluorescence by their different characteristics in the frequency or time domain have also been developed which are applicable even to opaque or inhomogeneous samples. Modulation of the detected optical frequency can separate broad fluorescence from sharper RS features. The more effective method of modulation of the excitation frequency distinguishes between fluorescence and RS in cases where the fluorescence spectrum is independent of excitation frequency, since the frequency of RS tracks the excitation frequency. This method fails, however, when a fraction of the fluorescence is emitted before the excitation is thermalized. In this case "hot luminescence" also tracks the exciting frequency to some extent. In addition, methods which exploit the different temporal response of RS and fluorescence to pulsed excitation have been developed to suppress fluorescence. Since RS is prompt, it is detected in a time which is the convolution of the laser pulse width and the detector response time. Fluorescence can be discriminated against if the detection window overlapping the laser probe is much shorter than the fluorescence decay time. Pulsed laser techniques are most effective in discriminating against fluorescence with lifetimes considerably greater than the response time of photomultiplier tubes of .gtoreq.0.3 nsec.
Another technique detects the modulated component of emission from a mechanically chopped laser beam using a lock-in detector following a photomultiplier tube. This method has been used to detect RS from ruby while suppressing fluorescence with a lifetime of about 5 msec. RS only occurs when the beam is chopped on while the depth of modulation of fluorescence is reduced when the period of chopping is less than 5 msec. The shortest luminescent lifetime that can be suppressed to some degree by this technique is limited by the chopping period of the laser beam, or the response time of the photomultiplier tube or lock-in detector.
The present invention includes the use of electro-optic demodulation of emission induced by an intensity modulated laser to null fluorescence. Phase sensitive detection can separate fluorescence from RS since the phase of the fluorescence lags the phase of the excitation, while RS, which is a prompt scattering process, is in phase with the excitation. Particularly when extended to higher frequencies the method of phase-resolved modulation RS can be used quite generally to suppress fluorescence even in samples with subnanosecond decay times as well as to measure ultrashort optical phenomena. Since fluorescence is suppressed by subtracting spectra in which fluorescence is present rather than by enhancing RS or rejecting fluorescence altogether, statistical noise arising from the fluorescence background is retained in the difference spectrum. This noise can be reduced by signal averaging.
It is demonstrated here that a phase resolved optical modulation approach can be used to observe RS in the presence of much more intense fluorescence. Using 30 MHz modulation, fluorescense with a lifetime of 8.5 nsec. is rejected. An extension of this approach to microwave frequencies will make it possible to suppress fluorescence with lifetimes as short as 50 psec. Shorter-lived species have low quantum yields for fluorescence and generally do not contribute significantly to fluorescence.